In this study, analysis of the mitochondrial and Y-chromosomal haplogroups was used to provide information about the phylogenetic groups of Tutankhamun’s family members, and their presence among the reported contemporary Egyptian population data. The mitochondrial and Y-chromosomal DNA analysis of Tutankhamun’s family confirms our previous data of the royal family pedigree, with multiple controls authenticating all results. The proposed sibling relationship between Tutankhamun’s parents, KV55 (Akhenaten) and KV35YL, is further supported. The royal lineage is composed of the Y-chromosome haplogroup R1b and the mitochondrial haplogroup K. Population genetics point to a common origin at ca. 14 000-28 000 years before present locating to the Near East.